Fluorescent proteins (FPs) have revolutionized biomedical research, but the full benefits of this technology have yet to be realized. Researchers have identified and engineered a variety of GFP-related proteins, including red FPs such as DsRed, but many of these new FPs give weaker fluorescence signals than GFP. Moreover, monomeric FPs often show a residual self- association that results in mislocalization and even toxicity of fluorescent fusion proteins. Until these problems are remedied, the new FPs will never be as broadly useful as GFP. The improvement of FPs will be aided by investigating the mechanisms of chromophore formation. Our goal is to devise robust directed evolution strategies for enhancing the brightness and minimizing the self-association of DsRed and other FPs. In parallel, structure-function analysis of wild-type and mutant FPs will help us to understand key properties such as the pathway of chromophore maturation. This project is a collaboration between two groups with complementary expertise. One PI (Glick) has extensively engineered DsRed, and has created rapidly maturing tetrameric and monomeric variants known commercially as "DsRed-Express" and "DsRed-Monomer", respectively. The other PI (Keenan) is a skilled structural biologist and an expert at improving proteins by directed evolution. This proposal has three Specific Aims. Specific Aim #1: To develop assays and procedures for generating brighter FPs that are less prone to self-association. We will use homology- and structure-guided mutagenesis to enhance the fluorescence signals from monomeric FPs, and to reduce the tendency of monomeric FPs to self-associate in vivo when present at high local protein concentrations. Specific Aim #2: To devise a systematic assay system for testing whether a monomeric FP is a suitable fusion tag. We will fuse a monomeric FP to a set of 96 potentially aggregation- sensitive partner proteins in yeast. The resulting in vivo fluorescence patterns will be subjected to detailed image analysis. Specific Aim #3: To characterize the chromophore maturation pathway and other properties of DsRed. The reactions that generate the DsRed chromophore are incompletely understood. We will test a novel maturation scheme that can explain previously puzzling results. Fluorescent proteins have become key experimental tools for basic and applied biomedical research. The studies described here will expand the applications of this technology.